In order to introduce new desirable characters into the cultivar assortment of lily a range of interspecific crossing barriers has to be overcome. By using various pollination and embryo rescue techniques pre- and postfertilization barriers were overcome and a range of wide interspecific lily hybrids between species and cultivars from the different sections of the genus Lilium could be made. Important breakthroughs include the development of the LA- (L. longiflorum x Asiatic hybrids), the LO- (L. longiflorum x Oriental hybrids) and the OA- (Oriental x Asiatic hybrids) hybrids. In general wide interspecific lily hybrids show F1-sterility. Using somatic chromosome doubling techniques (mitotic polyploidization) tetraploids with restored fertility can be produced from these diploid hybrids. An alternative method is the use of unreduced (or 2n) gametes (meiotic polyploidization), which are rarely found in some hybrids. Introgression of alien chromosome segments from donor species into recipient cultivar through backcrossing of F1 hybrid was studied using in situ hybridization techniques (GISH). Mitotic polyploidization showed no homoeologous recombinations between the parental genomes whereas meiotic polyploidization detected many. The use of 2n-gametes seems to be the most promising way for the introgression of desirable characters

Lim, K.B.

Maas, I.W.G.M.

Tuyl, J.M., van

English

Name not available

Introgression in interspecific hybrids of lily

Wageningen Yield 

  213
Introgression in Interspecific Hybrids of Lily 
 
Jaap M. van Tuyl, Ingrid W.G.M. Maas and Ki-Byung Lim  
Plant Research International 
Business Unit Genetics and Breeding  
P.O. Box 16, 6700 AA Wageningen,  
The Netherlands 
Tel:  +31-317-477329   
Fax: +31-317-418094 
E-mail J.M.vantuyl@plant.wag-ur.nl 
 
Keywords: Lilium, interspecific hybrids, introgression, meiotic, mitotic polyploidization, 
Genomic in situ hybridization (GISH). 
 
Abstract 
 In order to introduce new desirable characters into the cultivar assortment of 
lily a range of interspecific crossing barriers has to be overcome. By using various 
pollination and embryo rescue techniques pre- and postfertilization barriers were 
overcome and a range of wide interspecific lily hybrids between species and cultivars 
from the different sections of the genus Lilium could be made. Important 
breakthroughs include the development of the LA- (L. longiflorum ´  Asiatic hybrids), 
the LO- (L. longiflorum ´  Oriental hybrids) and the OA- (Oriental ´  Asiatic hybrids) 
hybrids. In general wide interspecific lily hybrids show F1-sterility. Using somatic 
chromosome doubling techniques (mitotic polyploidization) tetraploids with restored 
fertility can be produced from these diploid hybrids. An alternative method is the use of 
unreduced (or 2n) gametes (meiotic polyploidization), which are rarely found in some 
hybrids. Introgression of alien chromosome segments from donor species into recipient 
cultivar through backcrossing of F1 hybrid was studied using in situ hybridization 
techniques (GISH). Mitotic polyploidization showed no homoeologous recombinations 
between the parental genomes whereas meiotic polyploidization detected many. The 
use of 2n-gametes seems to be the most promising way for the introgression of 
desirable characters.  
 
INTRODUCTION 
 Interspecific crossing barriers need to be overcome in order to introduce new characters 
such as resistances, flower shape and colour, from wild species into the cultivar assortment of 
lily. Lily appeared to be a suitable model plant for studying interspecific crossing barriers. For 
overcoming pre- and post-fertilization barriers a range of techniques have been developed. 
Barriers that occur before fertilization, such as inhibition of pollentube growth in the style can be 
overcome by using pollination techniques, as the cut style method, the grafted-style method and 
the in vitro isolated ovule pollination technique (Asano & Myodo, 1977ab; Van Tuyl et al., 
1991).  
 Post-fertilization barriers can occur during stages of hybridization (e.g. firstly during the 
development of the hybrid embryo) and can be circumvented by in vitro pollination and/or 
rescue methods as embryo, ovary-slice and ovule culture (Asano, 1980; Van Tuyl et al., 1991; 
Okazaki et al. 1994). A second type of post-fertilization barrier can be found after raising the 
hybrid, manifesting itself in interspecific hybrids that show F1-sterility. Using mitotic 
(chromosome doubling) and meiotic polyploidization techniques amphidiploids with restored 
fertility can be produced from these diploid hybrids (Van Tuyl et al., 1992; Van Tuyl, 1993). 
The third post-fertilization barrier is a lack of introgression, caused by absence of recombination 
in cases when amphidiploid interspecific hybrids are used. Genomic in situ hybridization 
techniques offer an insight into the parental genome composition and homologous recombination 
breakpoints (Karlov et al., 1999, Lim et al., 2000). This paper describes some recent results of 
our interspecific hybridization research and the possibilities of introgression. 
 
Proc. 8th Int. Symp. on Flowerbulbs 
Eds. G. Littlejohn et al. 
Acta Hort. 570, ISHS 2002 
  214
Plant Material 
 Lilium hybrids and species which were used from the collection of Plant Research 
International (former CPRO-DLO) and are derived from different sections (between brackets) 
of the genus Lilium: L. longiflorum (Leucolirion), L. henryi (Leucolirion or Archelirion), L. 
canadense (Pseudolirium), L. bulbiferum, L. dauricum (Sinomartagon), L. candidum (Lilium), 
L. rubellum (Archelirion), L. martagon (Martagon), Asiatic hybrids  (Sinomartagon) and 
Oriental hybrids (Archelirion). 
Three diploid (2n=2x=24) 2n-gametes producing interspecific hybrids (LA) of L. longiflorum 
(L) ‘Gelria’ ´ Asiatic hybrid (A) ‘Whilito’ as well as their backcross progenies  (ALA) were 
used. All F1-hybrids were known to produce fertile 2n-pollen ranging from 0.1% up to 15%. 
The Asiatic hybrid ‘Montreux’ was used to produce backcross progenies.  
 Because F1-hybrids of L. longiflorum ‘Gelria’ ´ L. rubellum (LR) showed absolute 
sterility, mitotic polyploidization was performed by in vitro treatment of oryzalin. The selected 
amphidiploid (LLRR) plants resulting from artificial chromosome doubling were used as male 
parent in the back-cross with L. longiflorum ‘Snow Queen’ (2n=2x=24) and over a hundred 
BC1 plants were obtained (Lim et al. 2000). BC1 plants were selected and pollinated with 
tetraploid (LLLL) L. longiflorum to produce BC2 individuals.   
 
METHODS 
 
Pollination Methods:  
 The cut-style method (CSM) and the grafted style method (GSM) have been most 
frequently used. CSM: the style was cut with a razor blade 0-2 mm above the ovary, stigmatic 
fluid was applied followed by pollen. GSM: pollen was deposited on compatible stigma and 
after one day the style with germinating pollen was cut 1-2 mm above the ovary and attached to 
an ovary of the incongruent mother plant (Van Tuyl et al., 1991).  
 In vitro methods: Ovary culture, ovule and embryo culture  and chromosome doubling 
are applied as previously described (Van Tuyl et al., 1991, 1992).    
 Pollen tube penetration: Pollen tube penetration in the ovules: 5-10 days after pollination 
ovaries are used. One or two carpels, with the ovules attached to the placenta, are de-stained in 
a mixture of water, glycerol and lactic acid (1:2:1) and subsequently stained in a solution of 1% 
aniline blue in the same mixture before being  de-stained again. Penetration was observed using 
a light microscope (Janson et al., 1994). 
 Chromosome doubling: Mitotic chromosome doubling was carried out by treating in 
vitro bulb scales with 0.001% oryzalin and detecting by flowcytometric DNA-measurements 
(Van Tuyl et al. 1992; van Tuyl and Boon, 1997). 
 Chromosome preparation: Chromosomes were prepared by squashing fixed cells onto 
clean microscope slides. The method was modified from that of Karlov et al. (1999). Well-
squashed preparations were dipped for a few seconds in liquid nitrogen and, after taking off the 
cover slip, dehydrated in a graded ethanol series (70, 95, and 100%) and air dried.  The best 
slides were used for GISH-work. 
 DNA probes: Total genomic DNA was isolated from in vitro young leaves of L. 
longiflorum and labeled with digoxigenin-11-dUTP (Boehringer Mannheim) by nick translation 
according to the manufacturers instructions. Sheared herring sperm DNA was used for block 
DNA instead of L. rubellum DNA as a counterpart of L. longiflorum.  
 In situ hybridization: Genomic in situ hybridization was performed according to 
Khrustaleva and Kik (1998) with some modifications. Chromosome preparations were 
pretreated with RNase A (100 mg/mL) and pepsin (5 mg/mL), fixed with 4% r-formaldehyde 
for 10 minutes, dehydrated in an ethanol series and air dried. The hybridization mixture 
contained 50% deionized formamide, 10% (w/v) sodium dextran sulfate, 2´ SSC , 0.25% (w/v) 
SDS, 1.25-2.5 ng/ml genomic probe DNA, and 25 ng/ml herring sperm DNA. The mixtures 
were denatured for 10 min. at 70 ºC and then directly placed on ice for over 10 minutes. 44 mL 
of hybridization mixture was applied on each slide. Slides were denatured at 80 ºC for 10 
minutes and hybridised for overnight at 37 ºC in a humid chamber. Slides were washed for 15 
minutes in 2´  SSC, for 30 minutes in 0.1´ SSC at 42 ºC, followed by 5 minutes in buffer 1 (0.1 
  215
M Tris-Hcl with 0.15 M NaCl, pH 7.5). Digoxigenin-11-dUTP was detected with 20 mg/mL 
anti-Dig-FITC (fluorescein isothiocyanate; Boehringer Mannheim) and 20 mg/mL rabbit-anti-
sheep-FITC. Biotin-16-dUTP was detected with 4 mg/mL streptavidin-Cy3, 10 mg/mL 
biotinylated-antistreptavidin, and 4 mg/mL streptavidin-Cy3. The preparations were 
counterstained with 1 mg/mL DAPI and 4 mg/mL PI (propidium iodide). 
 
RESULTS AND DISCUSSION  
 In Figure 1 a simplified crossing polygon is presented of the genus Lilium, while a 
complete crossing polygon is published elsewhere (Van Tuyl et al. 2000). The most important 
crosses between L. longiflorum and the Asiatic hybrids and the Oriental hybrids are presented. 
The cross is unidirectional in case of the LA and the OA hybrids, but appears bi-directional in 
case of the LO-hybrids. More than 10.000 flowers were pollinated to produce these 3 groups 
of hybids. The LA-combination resulted in approximate of one hybrid per 2 pollinated flowers, 
the LO-combination one in four and the OA-combination one in ten. However, the percentage 
of hybrids per pollinated flower appeared to be genotype dependent in all cases. 
 The hybrids, obtained after intersectional crosses, are produced by using the cut- style 
method in combination with embryo culture or with ovary-slice and ovule culture. It has not been 
proven that by using the grafted style method other combinations can succeed inproducing other 
combinations than after using the cut style method, but the number of hybrid embryos obtained 
per ovary substantially increased in some combinations with the GSM-method. The grafted style 
method's problem, however, is that the pollen tubes are often not able to enter the style of the 
mother ovary because of inadequate attachment of the styles. 
 Restoration of F1-sterility by doubling the number of chromosomes was successfully 
performed in the following crosses: L. henryi x L. candidum, L. longiflorum x Asiatic hybrids, 
L. longiflorum x L. candidum, L. longiflorum x L. concolor, L. longiflorum x L. henryi, L. 
longiflorum x L. rubellum, L. longiflorum x Oriental hybrids, L. longiflorum x L. dauricum 
and Oriental x Asiatic hybrids. In all these F1-hybrids (except the OA-hybrids) the restoration 
was found in most autodiploids. Using these tetraploids backcrossings were performed on 
Asiatic and Oriental hybrids and L. longiflorum. An interesting backcross appeared to be L. 
longiflorum x (L. longiflorum x L. rubellum), resulting in a triploid population that showed all 
characters of a pink longiflorum. The characteristics were intermediate between L. longiflorum 
and L. rubellum. These triploids were backcrossed to L. longiflorum for introgression of the 
pink colour. An investigation into the possible introgression of characteristics from one species 
to the cultivated one was performed in the LR-hybrids by in situ hybridization techniques. A 
total absence of homoeologous recombinations was demonstrated in all 9 hybrids (Fig. 2b) (Lim 
et al. 2000). There was, however, a segregation of chromosomes in the BC2 (LLLR 2n=42, 
43, 44), in which 6, 7 or 8 chromosomes of L. rubellum were retained with three sets of L. 
longiflorum (Fig. 2c). In general it can be concluded that due to preferential pairing of the 
parental chromosomes (Fig. 2c) crossovers between parental chromosomes will not occur. 
 In case of the LA-hybrids, some fertility in a number of cases was found in the diploid 
F1's, caused by 2n-gametes occurring in the pollen or pollen mother cells. When investigating 
this material using GISH-techniques, there was clear evidence for homoeologus chromosome 
association and recombination at metaphase I and anaphase I stages of microsporogenesis (Fig. 
2d). The occurrence of such genetic recombination in LA-hybrids was also convincingly proven 
by the analysis of chromosome composition of BC1 (ALA) and BC2 progenies. (Fig. 2ef.) 
(Karlov et al. 1999; Lim et al. 2000). This recombination is highly important for introgression of 
specific characters from donor to recipient cultivars. It has been shown that by using 2n-
gametes, as in LA-hybrids, sexual polyploid progenies can be produced with the addition of 
complete alien chromosomes as well as recombinant chromosome segments of variable sizes. 
 In the near future polyploid Lilium-hybrids originated from a range of different 
genotypes (L. longiflorum, L. henryi, L. rubellum, Asiatic hybrids, Oriental hybrids), which 
previously could not be combined or recombined, will open up a whole range of new and 
promising possibilities for innovating the lily assortment.  
 
ACKNOWLEDGEMENTS 
  216
 The authors would like to thank the 11 Dutch lily breeding companies, who supported 
this research for many years. 
 
Literature Cited 
Asano, Y. 1980. Studies on crosses between distantly related species of lilies. IV. The culture 
of immature hybrid embryos 0.3 - 0.4 mm long. J. Japan. Soc. Hort. Sci. 49:114-118. 
Asano, Y. and Myodo, H. 1977a. Studies on crosses between distantly related species of lilies. 
I. For the intrastylar pollination technique. J. Japan. Soc. Hort. Sci. 46:59-65. 
Asano, Y. and Myodo, H. 1977b. Studies on crosses between distantly related species  of lilies. 
II. The culture of immature hybrid embryos. J. Japan. Soc. Hort. Sci. 46:267-273. 
Janson, J., Reinders, M.C., Valkering, A.G.M., Van Tuyl, J.M. and Keijzer, C.J. 1994. Pistil 
exudate production and pollen tube growth in Lilium longiflorum Thunb. Annals of Botany 
73:437-446. 
Karlov, G.I., Khrustaleva, L.I, Lim, K.B. and Van Tuyl, J.M. 1999. Homoeologous 
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genomic in situ hybridization (GISH). Genome  42(4):681-686. 
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CPRO-DLO.  The lily yearbook of the North American Lily Society 43:10-18. 
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Bino, R.J. 1991. Application of in vitro pollination, ovary culture, ovule culture and embryo 
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  217
Figurese 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig. 1. A simplified crossing polygon of the most important lily hybrid groups (L=L. 
longiflorum, A=Asiatic, O=Oriental) in the genus Lilium, resulting in wide 
interspecific hybrids (LA, LO and OA-hybrids). 
  218
 
Fig. 2. Genomic in situ hybridization of mitotic chromosomes of the F1, BC1 and BC2 plants of 
the LR- and the LA-hybrids. (a) The meiotic chromosomes of BC1 (LLR) with 12 
bivalents (yellow fluorescence) indicating L. longiflorum and 12 univalents (red 
fluorescence) representing L. rubellum. (b). 36 Chromosomes of the BC1 plant 
961003-27 without any recombinations. L. longiflorum (yellow fluorescence) and L 
rubellum (red fluorescence). (c) Aneuploid BC2 plant 982211-27 from backcrossing of 
the BC1 (LLR) to 4x L. longiflorum (LLLR). Thirty six (three sets) of L. longiflorum 
(yellow fluorescence) with 8 L. rubellum chromosomes (red fluorescence). (d) The 
meiotic chromosomes of LA 88542-52 in metaphase I with 2 bivalents indicating 
chromosome association of L. longiflorum (yellow) and Asiatic (red)  (e). 36 
Chromosomes of the ALA plant 921238-1 with three recombinations. L. longiflorum 
(yellow fluorescence) and L rubellum (blue fluorescence). (c) Aneuploid BC2 plant 
997139-2 (2n=30) from backcrossing of the BC1 (ALA)-hybrids to diploid Asiatic. 5 
chromosomes of L. longiflorum (green fluorescence) and 25 Asiatic chromosomes 
including 2 recombinant chromosomes. 


Introgression in interspecific hybrids of lily

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